The present invention relates generally to the field of diagnostic test compositions, and more particularly, to diagnostic tests useful for determination of creatinine and enzymatic products thereof.
Creatinine is a product of the endogenous metabolism of muscle. The amount of creatinine in the urine reflects total muscle mass and the degree of muscular activity. It is unrelated to dietary protein ingestion. It arises primarily from creatine and phosphocreatine during energyliberating processes in various body tissues, primarily muscle tissue.
The amount of creatinine in the urine of each individual is remarkably constant and varies little from day to day. Measurement of urinary creatinine may be used to determine the accuracy of 24-hour urine collections. Normal creatinine excretion ranges from 0.6 to 1.5 grams (g)/day in women and from 1.0 to 2.0 (g)/day for men. Creatine is almost completely reabsorbed by the renal tubules so that only a small amount, less than 100 milligrams (mg)/24 hours, is found in the urine. Therefore a principal value for the determination of creatinine is as a urine marker. In the majority of clinical conditions where urinalysis is part of a diagnostic profile, the creatinine level remains constant and there is an indicator of the volume and concentration or urine output in such patients.
Urinary creatine is elevated in the early stages of muscular dystrophy, when muscle destruction is occurring rapidly, and in any wasting disease involving increased tissue catabolism. It is elevated during severe and strenuous muscular activity and in hyperthyroidism. Urinary creatinine is decreased during the later stages of muscular dystrophy and whenever renal function is impaired. Urinary creatine increases in the same disease states that produce an increase in urinary creatinine.
The most widely known method for the determination of creatinine is the non-enzymatic Jaffe method which involves the formation of an orange-red color with an alkaline picrate solution. This method is not specific for creatinine, however, since many pseudocreatinine substances also react with alkaline picrate. Various attempts at improvement on the Jaffe method are discussed in Henry, Clinical Chemistry, at pages 541 et seq and in Dewhurst, U.S. Pat. No. 3,705,013. All are based on non-enzymatic color development for quantification of creatinine.
Many workers have sought a source of enzymes useful in creatinine determinations. Creatine and its anhydride, creatinine, do not occur in bacteria, yeast or molds. However, over forty different strains of bacteria and yeast have been shown to be capable of utilizing creatine and creatinine. The literature discloses the existence of at least four different enzymatic pathways whereby bacteria and molds may metabolize creatinine.
Appropriate soluble enzymes have therefore been sought which could catalyze specific, measurable reactions of creatinine. Among others, creatinase, creatininase, and a glycocyaminase have been characterized. An enzyme referred to as creatine-mutase has been isolated from soil bacteria and is responsible for the equilibrium adjustment between creatinine and creatine.
Mollering U.S. Pat. No. 3,806,416 discloses two enzymes referred to as creatinine amidohydrolase and creatine amidinohydrolase prepared from Alcaligenes spec. WS 51400 and Pencillium WS 90001. The former enzyme is disclosed to convert creatinine to creatine, and the latter, creatine to sarcosine plus urea. Holz Pat. No. 3,806,420 discloses a method for producing these enzymes from these strains.
Mollering U.S. Pat. No. 3,907,644 discloses a creatinine detection method wherein an aqueous creatinine-containing solution is incubated with creatinine amidohydrolase at a pH between 7.5 and 9 and either the creatine formed or the decrease of creatine is determined in known manner. Additionally, use of creatinase, also classified as creatine amidinohydrolase by the reference, to convert creatine into sarcosine and urea is disclosed. The sarcosine and urea can then be determined in conventional manner. A reagent combination is provided which includes a creatinine standard, picric acid, an aqueous solution of NaOH, and creatinine amidohydrolase alone or together with buffer and optionally in admixture with creatinase, in an unmixed state before use. Another reagent combination has a buffer, reduced nicotinamide-adeninedinucleotide (NADH), adenosine triphosphate (ATP) and phosphoenolpyruvate (PEP); lactate dehydrogenase (LDH), pyruvate kinase (PK) and magnesium chloride; (3) creatine kinase and (4) creatinine amidohydrolase.
Notwithstanding these efforts by prior workers in the field, there has remained the difficulty that the reagents necessary have not been compatible in a single aqueous solution. Further, tests have required caustic reagents, such as strong acids or bases. Thus it has heretofore been impossible to provide a unitized test composition or to incorporate the test reagents in a conveniently used format.
Also, prior tests have been designed for reading of ultraviolet light absorbance, thus requiring time consuming procedures and expensive equipment.